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Lek Pharmaceuticals, Drug Discovery, Ljubljana, Slovenia

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Presentation on theme: "Lek Pharmaceuticals, Drug Discovery, Ljubljana, Slovenia"— Presentation transcript:

1 Lek Pharmaceuticals, Drug Discovery, Ljubljana, Slovenia
STRUCTURAL PROPERTIES OF ANTIMICROBIAL PEPTIDES ACTING ON BACTERIAL MEMBRANES Boštjan Japelj Lek Pharmaceuticals, Drug Discovery, Ljubljana, Slovenia

2 Antibiotics – “miracle drugs”
Bacterial resistance is becoming a major problem in modern medicine

3 Cationic antimicrobial peptides:
- up to 50 aminoacids long a net positive charge of at least +2 (Arg, Lys) - antimicrobial activity against G-, G+ bacteria, fungi, protozoa, viruses, anticancer activity, effectors of innate immune response - 4 structural classes: ref:

4 - act on membranes and intracellular targets,
ref.: Matsuzaki, K.,. Biochim Biophys Acta, (1-2): p -advantage: fast acting, resistance is unlikely to develop, able to neutralize bacterial endotoxins and prevent development of sepsis

5 C12LF11 lauryl-FQWQRNIRKVR-NH2 P3-55 octanoyl-FWRIRIRR – NH2
LACTOFERRIN LF FQWQRNIRKVR-NH2 C12LF lauryl-FQWQRNIRKVR-NH2 P octanoyl-FWRIRIRR – NH2 Membrane models: LPS (lipopolysaccharide): model for baterial membrane SDS (sodium dodecyl sulphate): model for bacterial membrane DPC (dodecyl phosphocholine): model for eucaryotic membrane

6 NMR study of LF11 + S-LPS, LF11 + SDS, LF11 + DPC
LF11 + S-LPS: trNOE CPMG-T2 experiment: + LF11 LPS koff LF11-LPS TRNOE between aromatic and aliphatic side chains in 2 mM LF11 upon addition of 1/20 of molar ratio of LPS (b) and LTA (c). The reference NOESY spectrum of LF11 is shown in (a). Spectra were recorded at a mixing time of 150 ms.

7 LF11 + S-LPS Family of 3D structures of LF11 in complex with LPS.
( basic, hydrophobic, Complex between LF11 and LPS 441 Å2 of surface area buried polar residues)

8 Binding motif: LF11 :Phe1 , Arg5 , Lys9 , Arg11
Comparison of LPS interaction motifs in FhuA (left)1, LF11 (center)2 and polymyxin B (right)3,4 in the same orientation with respect to LPS, which would be in front of the plane of the page Binding motif: LF11 :Phe1 , Arg5 , Lys9 , Arg11 FhuA :Phe355, Lys439 , Arg384 , Lys351 PmxB :Phe6 , Dab8,9 , Dab3 , Dab1 1 Ferguson, A. D., Hofmann, E., Coulton, J. W., Diederichs, K., and Welte, W. (1998) Science 282: 2215–2220 2Japelj, B., Pristovšek, P., Majerle, A., Jerala, R. J Biol Chem, (17): p 3Pristovšek, P. and J. Kidrič, J Med Chem, (22): 4Pristovšek, P., Simčič, S., Wraber, B., Urleb, U. , J Med Chem, :

9 Comparison of structures LF11+S-LPS, LF11+SDS, LF11+DPC

10 N-terminal part of LF11 is protected from fluoresscence quenching
Fluorescence quenching LF11 FQWQRNIRKVR-NH2 F0, F…Fluorescence emission intensity in the absence and presence of the quencher(Q) [Q]… concentration of the quencher KSV… Stern-Volmer quenching constant

11 -acylation enhances antimicrobial activity against G- and G+ bacteria
C12LF11: -acylation enhances antimicrobial activity against G- and G+ bacteria -acylation stabilizes secondary structure CD spectra of LF11 and C12LF11 in DPC micelles Family of structures of C12LF11 in DPC1 1Japelj, B., Zorko, M., Majerle, A., Pristovšek, P.,et al.. JACS, (2007), 129:

12 OCTANOYL-F W R I R I R R – NH2
P3-55: OCTANOYL-F W R I R I R R – NH2

13 Circular dichroism spectra

14 Structure of P3-55 in DPC Structure of P3-55 in SDS Backbone conformation of P3-55 in DPC

15 Positioning and orientation of P3-55 in micelles
(NMR experiments using paramagnetic probes 5-DSA and 16-DSA) doxyl group

16 reference 5 - DSA NOESY TOCSY

17 Normalized I/Iref ratios of HN-Ha cross peaks in NOESY spectrum
P3-55 in SDS P3-55 in DPC

18 Molecular dynamics of P3-55 in DPC
Total energy (left) and temperature (right) of the system (P DPC SOL + 4 Cl-) during first 2 ns of simulation DPC DPC + P3-55 time [ps] * Ratios between princpal moments of inertia of DPC during simulation of P3-55 in DPC. Principal moments of inertia are shown in the table. *moments of inertia in units 104 amu nm2. Asymetry parameter, a, defined as a = (2I1-I2-I3)/(I1+I2+I3) Order parameter tensor elements of DPC micelle for the simulation of DPC micelle a) and DPC micelle + P3-55 b). –SCD=2/3Sxx + 1/2Syy Radial density of P3-55 in complex with DPC micelle. DPC coordinates were taken fromTieleman, D.P., et al. J. Phys Chem. B. 2000, 104:

19 Outer membrane Cytoplasmic membrane Mechanism of interaction of ANEPID peptides with the membrane of Gram-negative bacteria.

20 Acknowledgements: Andreja Majerle, Primož Pristovšek, Mateja Zorko, Roman Jerala (NIC, Ljubljana) Miha Kotnik, Katja Kristan, Drago Kuzman, Andrej Preželj, Jan Humljan, Petra Igličar, Vjekoslava Car, Uroš Urleb (Lek, Drug Discovery, Ljubljana) co-workers from the EU project ANEPID (Antimicrobial Endotoxin-neutralazing Peptides to Combat Infectious Deseases): Dagmar Zweytick, Karl Lohner (Graz) Guillermo Martinez de Tejada, Ignacio Moriyon, Susana Sanchez-Gomez (Pamplona) Sylvie E. Blondelle (San Diego, CA) Klaus Brandenburg, Jörg Andrä (Borstel)


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